Rapid kinetic measurements of ATP-dependent Ca++ accumulation and phosphoenzyme formation in sarcoplasmic reticulum membrane vesicles were carried out in order to determine the relationship between the conformational events associated with Ca++ translocation and the Ca++ ATPase intermediate reactions of the Ca++ pump reaction cycle. Results of these studies indicate that intravesicular Ca++ accumulation is delayed with respect to the appearance of a low affinity Ca++ binding site on the phosphorylated carrier. The properties of the low affinity Ca++ site are consistent with its involvement in Ca++ transport. The time dependence of these events suggest a translocation mechanism in which Ca++ is temporarily detained in compartment located between the outer and inner membrane surfaces following dissociation from the phosphorylated carrier and prior to release on the inside of the vesicle. The presence of a dimeric functional state was inferred from the behavior of the phosphorylation reactions coupled to Ca++ transport. The initial behavior of Na+ dependent Ca++ accumulation in cardiac sarcolemmal membrane vesicles was investigated by rapid quenching with EGTA. The time course exhibited an early burst phase which increased in the presence of inside positive charge and decreased when the inside became more negative with respect to the outside. The biphasic pattern of Ca++ uptake implies that a step or steps occurring subsequent to translocation is (are) rate-limiting in the reaction cycle. Modulation of the burst amplitude by changes in the transmembrane potential occurring during the cardiac action potential afford a possible mechanism for the rapid delivery of Ca++ to and removal of Ca++ from the myofilaments during the excitation-contraction cycle.